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A novel alkaline and low-temperature lipase ofBurkholderia cepacia isolated from Bohai in China for detergent formulation

A novel alkaline and low-temperature lipase ofBurkholderia cepacia isolated from Bohai in China... Annals of Microbiology, 59 (1) 105-110 (2009) A novel alkaline and low-temperature lipase of Burkholderia cepacia isolated from Bohai in China for detergent formulation HaiKuan WANG*, RuiJuan LIU, FuPing LU*, Wei QI, Jing SHAO, HuiJing MA Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, PO: 300457, P.R. China Received 17 October 2008 / Accepted 22 January 2009 Abstract - The bacterial strain LP08 was isolated from soil collected from bay of Bohai, China. The sequence of 16S rDNA of strain LP08 showed 99% homology to Burkholderia cepacia. The lipase from Burkholderia cepacia LP08 was purified by ammonium sulphate precipitation, ion exchange chromatography and Sephadex G-75 chromatography. The characterization of the lipase exhibited maximum activity at 30 °C and pH 9.0. The lipase retained 63, 66, 74, and 95% of its maximum activity at 10, 15, 20 and 25 °C respectively. The lipase activity was promoted in the presence of commercial detergent, sodium cholate, sodium taurocholate, glycerine and NaCl, while was little inhibited in the presence of TritonX-100, Tween-20, Tween-80, SDS, saponin. The present lipase was highly stable towards oxidizing agents and was stable after 1 h at 25 °C in the presence of hydrogen peroxide, sodium hypochlorite and sodium perborate. The results suggest that the lipase from Burkholderia cepacia LP08 showed good potential for application in the detergent formulation. Key words: Burkholderia cepacia; alkaline lipase; low-temperature lipase; purification; detergent. INTRODUCTION Low-temperature lipase might offer novel opportunities for biotechnological exploitation based on their high catalytic activ- Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) are one ity at low temperatures and unusual specificities. These proper- of the most important classes of hydrolytic enzymes that cata- ties are of interest in different fields such as detergents, textile lyze both the hydrolysis and the synthesis of esters (Sharma et and food industry, bioremediation and biocatalysis (Alquati et al., 2002). They are ubiquitous in nature and are produced by al., 2002). The most commercially important field of application various animals, plants, fungi and bacteria. Although a number for hydrolytic lipases is their addition to detergents, which are of lipase-producing bacterial sources are available, only a few used mainly in industrial laundry and in household dishwashers. are commercially exploited as wild or recombinant strains. Of The use of enzyme-based detergents is preferred over the con- these, the important ones are: Achromobacter, Alcaligenes, ventional synthetic ones due to their better cleaning properties, Arthrobacter, Bacillus, Burkholderia, Chromobacterium and lowering of washing temperatures and reduction in pollution. Pseudomonas (Gupta et al., 2004). Several kinds of lipases Lipases improve the washing capacity of detergents as well as originating from Burkholderia species have been identified and removal of fatty food stains and sebum from fabrics, which are their enzymatic properties and crystal structures have been difficult to remove under normal washing conditions (Hasan et elucidated (Rathi et al., 2001; Mandrich et al., 2005; Park et al., 2006; Saisubramanian et al., 2006). In 1994, Novo Nordisk al., 2007; Yang et al., 2007). Because of their preference for introduced the first commercial recombinant lipase ‘Lipolase’ the hydrolysis of triglycerides with a long chain length (great- which originated from the fungus Thermomyces lanuginosus er than C8), excellent enantioselectivity, transesterification, and was expressed in Aspergillus oryzae. In 1995, two bacte- esterification and tolerance to solvents and high temperature, rial lipases were introduced - ‘Lumafast’ from Pseudomonas Burkholderia lipases were extensively studied during the past mendocina and ‘Lipomax’ from Pseudomonas alcaligenes - by two decades for industrial use (Maury et al., 2005; Orcaire et Genencor International (Sharma et al., 2001). al., 2006; Fernades et al., 2007; Park et al., 2007; Yu et al., At present, lipases originated from Pseudomonas and 2007; Li et al., 2007). Burkholderia are most commonly used in household detergents (Park et al., 2007; Ruchi et al., 2007), but there is no report that Burkholderia produces both low-temperature and alkaline lipase. Here, we describe process for isolation of the strain LP08 * Corresponding Author. Phone: 86-22-60601958; producing both low-temperature and alkaline lipase and evalu- Fax: 86-22-60602298; E-mail: haikuanwangcn@yahoo.com.cn, ation of lipase as a detergent additive. lfp@tust.edu.cn 106 H. WANG et al. MATERIALS AND METHODS (Vorderwülbecke et al., 1992). Solution 1 contained pNPP (90 mg) dissolved in propane-2-ol (30 ml); solution 2 contained Isolation and screening of lipase-producing microorga- Triton X-100 (2 g) and gum arabic (0.5 g) dissolved in 450 -1 nisms. Three hundred and fifty-four of alkaline lipase-produc- ml buffer (Tris-HC1 50 mmol l , pH 8.0). The assay solution ing microorganisms were isolated from soil collected from bay was prepared by adding 1 ml of solution 1 to 9 ml of solution of Bohai, China with an olive oil alkaline plate, which contained 2 drop wise to get an emulsion which remained stable for 2 h. olive oil as the sole carbon source. Soil samples were inocu- The assay mixture contained 900 μl of the emulsion and 100 lated in 50 ml of enrichment medium, the medium contained: μl of the appropriately diluted lipase solution. The liberated -1 -1 -1 yeast extract 10 g l , K HPO 1 g l , MgSO ·7H O 2 g l , olive p-nitrophenol was measured at 410 nm. The molecular extinc- 2 4 4 2 -1 -1 -1 oil 20 g l , pH 9.5. The flasks were incubated at 26 °C for 3 tion coefficient of p-nitrophenol at 410 nm is 151 mmol cm . days under shaking 180 rpm. After inoculation, the culture One unit of lipase was defined as the amount of lipase that liquid was used for inoculation of another set of enrichment releases 1 mmol p-nitrophenol from the substrate for 1 min. flasks. The enriched culture was spread after serial dilution on screening medium. The screening medium contained: K HPO Purification procedure. 2 4 -1 -1 -1 1 g l , NaNO 3 g l , MgSO ·7H O 0.5 g l , FeSO ·7H O 0.01 Step 1: Solid ammonium sulphate was added to the superna- 3 4 2 4 2 -1 g l , emulsion of olive oil (it contained 0.2% Victoria blue B) 20 tant with stirring to bring the saturation to 35% and standing it -1 -1 g l , agar 20 g l , pH 9.5. The plates were incubated at 26 °C. at 4 °C for 4 h, the precipitate was removed by centrifugation Growing colonies with blue zones were isolated and transferred (10000 rpm at 4 °C for 20 min). Lipase activity both in precipi- -1 to slants, the slants contained: peptone 10 g l , yeast extract 5 tate and supernatant was determined. Additional ammonium -1 -1 g l , NaCl 10 g l , pH 7.5. The lipase activity was estimated by sulphate was added to the supernatant to bring the saturation plate assay method, as described below. By rough estimation, to 75% and the solution was left overnight. The precipitate was -1 the lipase from strain LP08 was optimal at low-temperature and collected and dissolved in 0.067 mol l phosphate buffer (pH alkaline range, so strain LP08 was chosen to use for following 8.0), then the solution was dialyzed against distilled water at experiments. 4 °C for 36 h. Bacterial strain identification. Identification of strain LP08 Step 2: The dialyzed solution was applied to a DE-52 column was conducted using 16S ribosomal DNA (rDNA) analysis (2.0 cm x 16 cm). The column was previously equilibrated with -1 (Eltaweel et al., 2005). The sequence analysis was performed three bed volumes of 0.067 mol l phosphate buffer (pH 8.0), by TaKaRa BioTechnology Corporation (Dalian, China). A and bound proteins were eluted with 60 ml linear NaCl gradient -1 homology search to reference strains registered in DDBJ/EMBL/ (0-1.0 mol l ) in the same buffer. The flow rate was adjusted to -1 GenBank was performed using NCBI BLAST. 15 ml h , the fraction volume of 3.5 ml was collected for lipase activity analysis. The active fractions were pooled and used for Lipase production. The seed inoculum was prepared by Sephadex G-75 column. inoculating a loop full of culture from a slant into 35 ml the seed -1 -1 medium (starch soluble 10 g l , bean flour 20 g l , corn syrup Step 3: The fraction containing lipase was chromatographed -1 -1 20 g l , K HPO 1 g l ) and incubated for 12 h at 28 °C. Two on Sephadex G-75 column (1.6 cm x 80 cm) equilibrated with 2 4 -1 millilitres were inoculated in 250 ml flask containing 35 ml of 0.067 mol l phosphate buffer (pH 8.0) and then the lipase fermentation medium with following composition: starch solu- was eluted with the same buffer. The flow rate was adjusted -1 -1 -1 -1 ble 10 g l , bean flour 20 g l , corn syrup 20 g l , K HPO 1 to 42.0 ml h and then the fraction volume of 3.5 ml was col- 2 4 -1 -1 g l , emulsion of bean oil 20 g l , the initial pH of the medium lected for lipase activity analysis. SDS-PAGE was used to prove was adjusted to pH 9.0, then incubated at 28 °C for 44 h under the purity of the lipase samples. shaking condition (180 rpm). After the incubation period, the cell-free supernatant was obtained by centrifugation at 10000 Lipase characterization. The effect of temperature on lipase rpm at 4 °C for 20 min. The supernatant was considered as activity was studied by carrying out the lipase reaction at dif- crude enzyme. ferent temperatures in the range of 10-60 °C at pH 8.0 using -1 0.067 mol l phosphate buffer. For studying thermal stability, -1 Assay of lipase activity. Lipase activity was determined by 1 ml of lipase was mixed with 19 ml of 0.067 mol l phosphate following two methods. buffer (pH 8.0) and incubated at 25, 30, 35 and 40°C for 84 h. Residual lipase activity was determined at different intervals Plate assay method. The underlying principle employed in this from 0 to 84 h, at pH 8.0 and 25 °C. method was based on change of the indicator (Victoria blue) For the determination of the effect of pH on lipase, lipase caused by free fatty acids liberated from olive oil under proper activity was measured at 25 °C in a pH range of 5.0-10.6 using -1 conditions. The medium containing 10% emulsion of olive oil (it different buffers, 0.067 mol l phosphate buffer (pH 5.0-9.0) -1 contained 0.2% Victoria blue B). The medium was adjusted to and 0.05 mol l glycine-NaOH buffer (pH 9.0-10.6). For pH -1 different pH using buffers: 0.067 mol l phosphate buffer (pH stability studies, 1 ml of lipase was mixed with 19 ml of buffers -1 5.0-9.0) and 0.05 mol l glycine-NaOH buffer (pH 9.0-10.6). with different pH (5.0-9.0) and incubated at 25 °C for 84 h. To quantify lipase activity 4-mm-diameter holes were punched Subsequently, the residual enzymatic activity was determined into the agar and filled with 20 μl of cell-free culture superna- by lipase activity assay. tant. The plate were incubated for 24 h at different temperature (15, 25, 35, 45 and 55 °C). Lipase activity was assayed by Evaluation of lipase as an additive for detergent formula- determined change of the blue zones. tion. Lipase from strain LP08 was biochemically characterized for its potential application in the detergent industry. The lipase sam- Spectrophotometric method. Spectrophotometric meth- ple was incubated in presence of surfactants viz. Triton X-100, od utilized p-nitrophenyl palmitate (pNPP) as substrate Tween-20, Tween-80, SDS, sodium cholate, sodium taurocholate, Ann. Microbiol., 59 (1), 105-110 (2009) 107 commercial detergents, glycerine, NaCl, Borax and sodium cit- rate at different concentration at 25 °C for 1 h and lipase activity was determined at pH 8.0 and 30 °C. Lipase stability in the presence of hydrogen peroxide, sodium perborate and sodium hypochlorite at 1% (w/v or v/v) at 25 °C for 1 h was checked and lipase activity was determined at pH 8.0 and 30 °C. AB C FIG. 1 - The size of rings formed on Victoria blue agar plate with the crude lipase of four different strains. In the three RESULTS plates, 2 is lipase from Burkholderia cepacia LP08, 1, 3 and 4 are lipase from other strains. A: The lipase reac- Screening of lipase-producing bacterial strains tion at 25 °C, pH 8.0. B: The lipase reaction at 25 °C, A total of 354 bacterial isolates from soil were screened for pro- pH 9.0. C: The lipase reaction at 35 °C, pH 9.0. ducing lipase, among which 51 isolates were obtained based on the lipase activity, while the others were discarded based on their comparatively poor lipase activity. Of the 51 isolates, the strain LP08 had the maximum lipase activity at 25 °C and pH 9.0 (Fig. 1). The 16S rDNA sequence of strain LP08 was analysed with GenBank database, and the sequence showed 99% homology to Burkholderia cepacia. Purification of lipase The lipase from Burkholderia cepacia LP08 was purified employ- ing a three-step procedure (Table 1). A 23.79-fold purification was obtained with a recovery of 13%. The specific activity of the -1 purified enzyme was 58.53 U mg of protein. Coomassie Brilliant Blue staining revealed the presence of a single protein with a molecular weight of 39 kDa (Fig. 2). Characterization of lipase As show in Fig. 3, the optimum temperature of the lipase from FIG. 2 - SDS-PAGE analysis of lipase from Burkholderia cepa- Burkholderia cepacia LP08 was 30 °C. The lipase retained 63, cia LP08 at various stages of purification. Lane 1: ion 66, 74, and 95% of its maximum activity at 10, 15, 20 and 25 exchange, lane 2: gel filtration, lane M: molecular °C respectively. The lipase retained more than 89 and 50% of its weight markers. activity for 36 and 84 h respectively at 30 °C, and the half life of the lipase was 75, 72, 52 h at 25, 35 and 40 °C respectively (Fig. 4) The lipase from Burkholderia cepacia LP08 showed activity in a very wide pH range (5.0-10.6). Maximal activity (100%) was observed at pH 9.0 (Fig. 5), while this was closely followed by pH 8.0 and pH 10.0 (97% and 91% of the maximum). The activity reduced drastically at pH 7.0 (84%) and was 33% of the maximum at pH 5.0. The lipase showed good stability after 84 h in an alkaline pH range, where the lipase retained 55 and 42% of the maximum activity at pH 9.0 and pH 8.0 respectively, but it was totally inactivated at acidic pH and lost 94% of activity at pH 5.0 (Fig. 6). Lipase stability towards surfactants, detergents, oxidizing agents and proteases Results presented in Table 2 reveal that the lipase was stable in FIG. 3 - Effect of temperature on the activity of lipase from some surfactants and retained 90.3, 91.6, 93.3, 84 and 60.4% Burkholderia cepacia LP08. TABLE 1 - Purification of the lipase from Burkholderia cepacia LP08 Purification Step Total activity Total protein Specific activity Activity yield Protein yield Purification -1 (U) (mg) (U mg ) (%) (%) (fold) Crude enzyme 765 311 2.46 100 100 1.00 (cell free supernatant) Precipitation 689 84 8.20 90 27 3.33 (NH ) SO (30-70%) 4 2 4 Ion exchange 275 11.3 24.34 36 3.6 9.89 Gel filtration 99.5 1.7 58.53 13 0.5 23.79 108 H. WANG et al. of its control activity in the presence of Triton X-100, Tween-20, Tween-80, saponin and SDS respectively. Lipase shows higher activity than the control in the presence of sodium cholate, sodium taurocholate, glycerine, sodium citrate and NaCl, as it retained 131.5, 177.9, 281, 266 and 167% of the control activity. Interestingly, the present lipase is highly stable towards oxidiz- ing agents and was stable after 1 h at 25 °C in the presence of hydrogen peroxide, sodium hypochlorite and sodium perborate. Alkaline protease (0.01%) had no effect on the lipase activity. The effect on the lipase activity of Borax has relation to the solu- tion concentration: low concentrations increased the lipase activ- ity while high concentrations inhibited the lipase activity. DISCUSSION Lipase used in detergents needs to be stable under alkaline pH and should be active in the presence of surfactants, bleaching FIG. 4 - Thermostability of lipase from Burkholderia cepacia agents and detergents (Sharma et al., 2001, 2002) LP08. A series of studies on characterization of lipase indicated that the optimal temperature and pH of lipase from Burkholderia cepacia LP08 are in low-temperature and alkaline range. At present, lots of reports show that lipases from Burkholderia and Pseudomonas has their optimum activity at high temperature and in alkaline range. Rathi et al. (2000, 2001) reported that optimum activity of lipase from Burkholderia cepacia was at 90 °C and at pH 11. Yang et al. (2007) found that the optimal tem- perature 70 °C and pH 8.0 for lipase from Burkholderia cepacia strain G63, and kept stable at a temperature range of 40-70 °C, after incubation at 70 °C for 10 h it remained 86.1% of its activity. Liu et al. (2006) reported that the optimal reaction con- ditions of lipase from Burkholderia sp. C20 were pH 9.0 and 55 °C. Park et al. (2007) reported that lipase from Burkholderia sp. HY-10 exhibited highest activities at 60 °C and pH 8.5. The lipase from Pseudomonas aeruginosa LP602 exhibited maximum lipase activity at pH 8.0 where it was also stably maintained. At 55 °C, the lipase had the highest activity but not stability (Dharmsthiti and Kuhasuntisuk, 1998). Umesh et al. (2003) reported that lipase from Pseudomonas mendocina PK-12CS was stable at room temperature for more than a month and expressed maxi- FIG. 5 - Effect of pH on the activity of lipase from Burkholderia mum activity at 37 °C and pH 8.0. Gao et al. (2000) reported cepacia LP08. that lipase from Pseudomonas had maximum activity at 45 °C and pH 9.0. Kulkarniand Gadre (2002) reported that lipase from Pseudomonas fluorescens NS2W had an optimal activity at 55 °C and at pH 9.0. Lin et al. (1996) reported that lipase from Pseudomonas pseudoalcaligenes F-111 was stable in the pH range of 6 to 10, and exhibited highest activities at 40 °C. There is no report about Burkholderia produces the low-temperature and alkaline lipase as we know so far. Besides optimal temperature and pH are in low-temperature and alkaline range, a good detergent lipase should also be sta- ble in the presence of surfactants. The lipase from Burkholderia cepacia LP08 was stable in the presence of some surfactants. The above finding was similar to that by Umesh et al. (2003) who found that lipase from Pseudomonas mendocina PK-12CS showed appreciably good stability to Triton X-100, Tween-20 and Tween-80. Ruchi et al. (2007) reported that lipase from Pseudomonas aeruginosa retained 100% activity in presence of Triton X-100, Tween-20 and Tween-80. Rathi et al. (2001) reported that the lipase from Burkholderia cepacia retained 93, 57, 40 and 53% of the activity in the presence of Triton X-100, Tween-20, Tween-80 and saponin respectively. On the other hand, Karadzic et al. (2006) reported lipase from Pseudomonas FIG. 6 - pH stability of lipase from Burkholderia cepacia LP08. aeruginosa was strongly stimulated by Triton X-100 and Tween- Ann. Microbiol., 59 (1), 105-110 (2009) 109 TABLE 2 - Lipase stability in presence of the surfactants, component of detergents, oxidizing agents and protease Surfactants / Protease Relative Detergents/Oxidizing agents Relative (1% w/v or v/v) activity (%) (1% w/v or v/v) activity (%) Control 100 Control 100 Surfactants 1% TritonX-100 69 Component of 4% Glycerine 209 detergents 0.1% TritonX-100 90.3 0.4% Glycerine 281 1% Tween-20 52.9 4% NaCl 266 0.1% Tween-20 91.6 0.4% NaCl 244 1% Tween-80 58.4 3% Borax 77.4 0.1% Tween-80 93.3 0.3% Borax 287 1% SDS 18.5 2% Sodium citrate 116 0.1% SDS 60.4 0.2% Sodium citrate 167 1% Saponin 53 Oxidizing agents 1% Hydrogen peroxide 91 0.1% Saponin 84 0.1% Hydrogen peroxide 93 1% Sodium cholate 131.5 1% Sodium perborate 95 0.1% Sodium cholate 125.2 0.1% Sodium perborate 96 1% Sodium taurocholate 177.9 1% Sodium hypochlorite 92 0.1% Sodium taurocholate 138.3 0.1% Sodium hypochlorite 95 Alkaline protease 0.1% 88.4 Commercial 1% 134 detergent (The 0.01% 100 0.1% 104 brand of bilang) 80. 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A novel alkaline and low-temperature lipase ofBurkholderia cepacia isolated from Bohai in China for detergent formulation

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Springer Journals
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Copyright © 2009 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 59 (1) 105-110 (2009) A novel alkaline and low-temperature lipase of Burkholderia cepacia isolated from Bohai in China for detergent formulation HaiKuan WANG*, RuiJuan LIU, FuPing LU*, Wei QI, Jing SHAO, HuiJing MA Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, PO: 300457, P.R. China Received 17 October 2008 / Accepted 22 January 2009 Abstract - The bacterial strain LP08 was isolated from soil collected from bay of Bohai, China. The sequence of 16S rDNA of strain LP08 showed 99% homology to Burkholderia cepacia. The lipase from Burkholderia cepacia LP08 was purified by ammonium sulphate precipitation, ion exchange chromatography and Sephadex G-75 chromatography. The characterization of the lipase exhibited maximum activity at 30 °C and pH 9.0. The lipase retained 63, 66, 74, and 95% of its maximum activity at 10, 15, 20 and 25 °C respectively. The lipase activity was promoted in the presence of commercial detergent, sodium cholate, sodium taurocholate, glycerine and NaCl, while was little inhibited in the presence of TritonX-100, Tween-20, Tween-80, SDS, saponin. The present lipase was highly stable towards oxidizing agents and was stable after 1 h at 25 °C in the presence of hydrogen peroxide, sodium hypochlorite and sodium perborate. The results suggest that the lipase from Burkholderia cepacia LP08 showed good potential for application in the detergent formulation. Key words: Burkholderia cepacia; alkaline lipase; low-temperature lipase; purification; detergent. INTRODUCTION Low-temperature lipase might offer novel opportunities for biotechnological exploitation based on their high catalytic activ- Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) are one ity at low temperatures and unusual specificities. These proper- of the most important classes of hydrolytic enzymes that cata- ties are of interest in different fields such as detergents, textile lyze both the hydrolysis and the synthesis of esters (Sharma et and food industry, bioremediation and biocatalysis (Alquati et al., 2002). They are ubiquitous in nature and are produced by al., 2002). The most commercially important field of application various animals, plants, fungi and bacteria. Although a number for hydrolytic lipases is their addition to detergents, which are of lipase-producing bacterial sources are available, only a few used mainly in industrial laundry and in household dishwashers. are commercially exploited as wild or recombinant strains. Of The use of enzyme-based detergents is preferred over the con- these, the important ones are: Achromobacter, Alcaligenes, ventional synthetic ones due to their better cleaning properties, Arthrobacter, Bacillus, Burkholderia, Chromobacterium and lowering of washing temperatures and reduction in pollution. Pseudomonas (Gupta et al., 2004). Several kinds of lipases Lipases improve the washing capacity of detergents as well as originating from Burkholderia species have been identified and removal of fatty food stains and sebum from fabrics, which are their enzymatic properties and crystal structures have been difficult to remove under normal washing conditions (Hasan et elucidated (Rathi et al., 2001; Mandrich et al., 2005; Park et al., 2006; Saisubramanian et al., 2006). In 1994, Novo Nordisk al., 2007; Yang et al., 2007). Because of their preference for introduced the first commercial recombinant lipase ‘Lipolase’ the hydrolysis of triglycerides with a long chain length (great- which originated from the fungus Thermomyces lanuginosus er than C8), excellent enantioselectivity, transesterification, and was expressed in Aspergillus oryzae. In 1995, two bacte- esterification and tolerance to solvents and high temperature, rial lipases were introduced - ‘Lumafast’ from Pseudomonas Burkholderia lipases were extensively studied during the past mendocina and ‘Lipomax’ from Pseudomonas alcaligenes - by two decades for industrial use (Maury et al., 2005; Orcaire et Genencor International (Sharma et al., 2001). al., 2006; Fernades et al., 2007; Park et al., 2007; Yu et al., At present, lipases originated from Pseudomonas and 2007; Li et al., 2007). Burkholderia are most commonly used in household detergents (Park et al., 2007; Ruchi et al., 2007), but there is no report that Burkholderia produces both low-temperature and alkaline lipase. Here, we describe process for isolation of the strain LP08 * Corresponding Author. Phone: 86-22-60601958; producing both low-temperature and alkaline lipase and evalu- Fax: 86-22-60602298; E-mail: haikuanwangcn@yahoo.com.cn, ation of lipase as a detergent additive. lfp@tust.edu.cn 106 H. WANG et al. MATERIALS AND METHODS (Vorderwülbecke et al., 1992). Solution 1 contained pNPP (90 mg) dissolved in propane-2-ol (30 ml); solution 2 contained Isolation and screening of lipase-producing microorga- Triton X-100 (2 g) and gum arabic (0.5 g) dissolved in 450 -1 nisms. Three hundred and fifty-four of alkaline lipase-produc- ml buffer (Tris-HC1 50 mmol l , pH 8.0). The assay solution ing microorganisms were isolated from soil collected from bay was prepared by adding 1 ml of solution 1 to 9 ml of solution of Bohai, China with an olive oil alkaline plate, which contained 2 drop wise to get an emulsion which remained stable for 2 h. olive oil as the sole carbon source. Soil samples were inocu- The assay mixture contained 900 μl of the emulsion and 100 lated in 50 ml of enrichment medium, the medium contained: μl of the appropriately diluted lipase solution. The liberated -1 -1 -1 yeast extract 10 g l , K HPO 1 g l , MgSO ·7H O 2 g l , olive p-nitrophenol was measured at 410 nm. The molecular extinc- 2 4 4 2 -1 -1 -1 oil 20 g l , pH 9.5. The flasks were incubated at 26 °C for 3 tion coefficient of p-nitrophenol at 410 nm is 151 mmol cm . days under shaking 180 rpm. After inoculation, the culture One unit of lipase was defined as the amount of lipase that liquid was used for inoculation of another set of enrichment releases 1 mmol p-nitrophenol from the substrate for 1 min. flasks. The enriched culture was spread after serial dilution on screening medium. The screening medium contained: K HPO Purification procedure. 2 4 -1 -1 -1 1 g l , NaNO 3 g l , MgSO ·7H O 0.5 g l , FeSO ·7H O 0.01 Step 1: Solid ammonium sulphate was added to the superna- 3 4 2 4 2 -1 g l , emulsion of olive oil (it contained 0.2% Victoria blue B) 20 tant with stirring to bring the saturation to 35% and standing it -1 -1 g l , agar 20 g l , pH 9.5. The plates were incubated at 26 °C. at 4 °C for 4 h, the precipitate was removed by centrifugation Growing colonies with blue zones were isolated and transferred (10000 rpm at 4 °C for 20 min). Lipase activity both in precipi- -1 to slants, the slants contained: peptone 10 g l , yeast extract 5 tate and supernatant was determined. Additional ammonium -1 -1 g l , NaCl 10 g l , pH 7.5. The lipase activity was estimated by sulphate was added to the supernatant to bring the saturation plate assay method, as described below. By rough estimation, to 75% and the solution was left overnight. The precipitate was -1 the lipase from strain LP08 was optimal at low-temperature and collected and dissolved in 0.067 mol l phosphate buffer (pH alkaline range, so strain LP08 was chosen to use for following 8.0), then the solution was dialyzed against distilled water at experiments. 4 °C for 36 h. Bacterial strain identification. Identification of strain LP08 Step 2: The dialyzed solution was applied to a DE-52 column was conducted using 16S ribosomal DNA (rDNA) analysis (2.0 cm x 16 cm). The column was previously equilibrated with -1 (Eltaweel et al., 2005). The sequence analysis was performed three bed volumes of 0.067 mol l phosphate buffer (pH 8.0), by TaKaRa BioTechnology Corporation (Dalian, China). A and bound proteins were eluted with 60 ml linear NaCl gradient -1 homology search to reference strains registered in DDBJ/EMBL/ (0-1.0 mol l ) in the same buffer. The flow rate was adjusted to -1 GenBank was performed using NCBI BLAST. 15 ml h , the fraction volume of 3.5 ml was collected for lipase activity analysis. The active fractions were pooled and used for Lipase production. The seed inoculum was prepared by Sephadex G-75 column. inoculating a loop full of culture from a slant into 35 ml the seed -1 -1 medium (starch soluble 10 g l , bean flour 20 g l , corn syrup Step 3: The fraction containing lipase was chromatographed -1 -1 20 g l , K HPO 1 g l ) and incubated for 12 h at 28 °C. Two on Sephadex G-75 column (1.6 cm x 80 cm) equilibrated with 2 4 -1 millilitres were inoculated in 250 ml flask containing 35 ml of 0.067 mol l phosphate buffer (pH 8.0) and then the lipase fermentation medium with following composition: starch solu- was eluted with the same buffer. The flow rate was adjusted -1 -1 -1 -1 ble 10 g l , bean flour 20 g l , corn syrup 20 g l , K HPO 1 to 42.0 ml h and then the fraction volume of 3.5 ml was col- 2 4 -1 -1 g l , emulsion of bean oil 20 g l , the initial pH of the medium lected for lipase activity analysis. SDS-PAGE was used to prove was adjusted to pH 9.0, then incubated at 28 °C for 44 h under the purity of the lipase samples. shaking condition (180 rpm). After the incubation period, the cell-free supernatant was obtained by centrifugation at 10000 Lipase characterization. The effect of temperature on lipase rpm at 4 °C for 20 min. The supernatant was considered as activity was studied by carrying out the lipase reaction at dif- crude enzyme. ferent temperatures in the range of 10-60 °C at pH 8.0 using -1 0.067 mol l phosphate buffer. For studying thermal stability, -1 Assay of lipase activity. Lipase activity was determined by 1 ml of lipase was mixed with 19 ml of 0.067 mol l phosphate following two methods. buffer (pH 8.0) and incubated at 25, 30, 35 and 40°C for 84 h. Residual lipase activity was determined at different intervals Plate assay method. The underlying principle employed in this from 0 to 84 h, at pH 8.0 and 25 °C. method was based on change of the indicator (Victoria blue) For the determination of the effect of pH on lipase, lipase caused by free fatty acids liberated from olive oil under proper activity was measured at 25 °C in a pH range of 5.0-10.6 using -1 conditions. The medium containing 10% emulsion of olive oil (it different buffers, 0.067 mol l phosphate buffer (pH 5.0-9.0) -1 contained 0.2% Victoria blue B). The medium was adjusted to and 0.05 mol l glycine-NaOH buffer (pH 9.0-10.6). For pH -1 different pH using buffers: 0.067 mol l phosphate buffer (pH stability studies, 1 ml of lipase was mixed with 19 ml of buffers -1 5.0-9.0) and 0.05 mol l glycine-NaOH buffer (pH 9.0-10.6). with different pH (5.0-9.0) and incubated at 25 °C for 84 h. To quantify lipase activity 4-mm-diameter holes were punched Subsequently, the residual enzymatic activity was determined into the agar and filled with 20 μl of cell-free culture superna- by lipase activity assay. tant. The plate were incubated for 24 h at different temperature (15, 25, 35, 45 and 55 °C). Lipase activity was assayed by Evaluation of lipase as an additive for detergent formula- determined change of the blue zones. tion. Lipase from strain LP08 was biochemically characterized for its potential application in the detergent industry. The lipase sam- Spectrophotometric method. Spectrophotometric meth- ple was incubated in presence of surfactants viz. Triton X-100, od utilized p-nitrophenyl palmitate (pNPP) as substrate Tween-20, Tween-80, SDS, sodium cholate, sodium taurocholate, Ann. Microbiol., 59 (1), 105-110 (2009) 107 commercial detergents, glycerine, NaCl, Borax and sodium cit- rate at different concentration at 25 °C for 1 h and lipase activity was determined at pH 8.0 and 30 °C. Lipase stability in the presence of hydrogen peroxide, sodium perborate and sodium hypochlorite at 1% (w/v or v/v) at 25 °C for 1 h was checked and lipase activity was determined at pH 8.0 and 30 °C. AB C FIG. 1 - The size of rings formed on Victoria blue agar plate with the crude lipase of four different strains. In the three RESULTS plates, 2 is lipase from Burkholderia cepacia LP08, 1, 3 and 4 are lipase from other strains. A: The lipase reac- Screening of lipase-producing bacterial strains tion at 25 °C, pH 8.0. B: The lipase reaction at 25 °C, A total of 354 bacterial isolates from soil were screened for pro- pH 9.0. C: The lipase reaction at 35 °C, pH 9.0. ducing lipase, among which 51 isolates were obtained based on the lipase activity, while the others were discarded based on their comparatively poor lipase activity. Of the 51 isolates, the strain LP08 had the maximum lipase activity at 25 °C and pH 9.0 (Fig. 1). The 16S rDNA sequence of strain LP08 was analysed with GenBank database, and the sequence showed 99% homology to Burkholderia cepacia. Purification of lipase The lipase from Burkholderia cepacia LP08 was purified employ- ing a three-step procedure (Table 1). A 23.79-fold purification was obtained with a recovery of 13%. The specific activity of the -1 purified enzyme was 58.53 U mg of protein. Coomassie Brilliant Blue staining revealed the presence of a single protein with a molecular weight of 39 kDa (Fig. 2). Characterization of lipase As show in Fig. 3, the optimum temperature of the lipase from FIG. 2 - SDS-PAGE analysis of lipase from Burkholderia cepa- Burkholderia cepacia LP08 was 30 °C. The lipase retained 63, cia LP08 at various stages of purification. Lane 1: ion 66, 74, and 95% of its maximum activity at 10, 15, 20 and 25 exchange, lane 2: gel filtration, lane M: molecular °C respectively. The lipase retained more than 89 and 50% of its weight markers. activity for 36 and 84 h respectively at 30 °C, and the half life of the lipase was 75, 72, 52 h at 25, 35 and 40 °C respectively (Fig. 4) The lipase from Burkholderia cepacia LP08 showed activity in a very wide pH range (5.0-10.6). Maximal activity (100%) was observed at pH 9.0 (Fig. 5), while this was closely followed by pH 8.0 and pH 10.0 (97% and 91% of the maximum). The activity reduced drastically at pH 7.0 (84%) and was 33% of the maximum at pH 5.0. The lipase showed good stability after 84 h in an alkaline pH range, where the lipase retained 55 and 42% of the maximum activity at pH 9.0 and pH 8.0 respectively, but it was totally inactivated at acidic pH and lost 94% of activity at pH 5.0 (Fig. 6). Lipase stability towards surfactants, detergents, oxidizing agents and proteases Results presented in Table 2 reveal that the lipase was stable in FIG. 3 - Effect of temperature on the activity of lipase from some surfactants and retained 90.3, 91.6, 93.3, 84 and 60.4% Burkholderia cepacia LP08. TABLE 1 - Purification of the lipase from Burkholderia cepacia LP08 Purification Step Total activity Total protein Specific activity Activity yield Protein yield Purification -1 (U) (mg) (U mg ) (%) (%) (fold) Crude enzyme 765 311 2.46 100 100 1.00 (cell free supernatant) Precipitation 689 84 8.20 90 27 3.33 (NH ) SO (30-70%) 4 2 4 Ion exchange 275 11.3 24.34 36 3.6 9.89 Gel filtration 99.5 1.7 58.53 13 0.5 23.79 108 H. WANG et al. of its control activity in the presence of Triton X-100, Tween-20, Tween-80, saponin and SDS respectively. Lipase shows higher activity than the control in the presence of sodium cholate, sodium taurocholate, glycerine, sodium citrate and NaCl, as it retained 131.5, 177.9, 281, 266 and 167% of the control activity. Interestingly, the present lipase is highly stable towards oxidiz- ing agents and was stable after 1 h at 25 °C in the presence of hydrogen peroxide, sodium hypochlorite and sodium perborate. Alkaline protease (0.01%) had no effect on the lipase activity. The effect on the lipase activity of Borax has relation to the solu- tion concentration: low concentrations increased the lipase activ- ity while high concentrations inhibited the lipase activity. DISCUSSION Lipase used in detergents needs to be stable under alkaline pH and should be active in the presence of surfactants, bleaching FIG. 4 - Thermostability of lipase from Burkholderia cepacia agents and detergents (Sharma et al., 2001, 2002) LP08. A series of studies on characterization of lipase indicated that the optimal temperature and pH of lipase from Burkholderia cepacia LP08 are in low-temperature and alkaline range. At present, lots of reports show that lipases from Burkholderia and Pseudomonas has their optimum activity at high temperature and in alkaline range. Rathi et al. (2000, 2001) reported that optimum activity of lipase from Burkholderia cepacia was at 90 °C and at pH 11. Yang et al. (2007) found that the optimal tem- perature 70 °C and pH 8.0 for lipase from Burkholderia cepacia strain G63, and kept stable at a temperature range of 40-70 °C, after incubation at 70 °C for 10 h it remained 86.1% of its activity. Liu et al. (2006) reported that the optimal reaction con- ditions of lipase from Burkholderia sp. C20 were pH 9.0 and 55 °C. Park et al. (2007) reported that lipase from Burkholderia sp. HY-10 exhibited highest activities at 60 °C and pH 8.5. The lipase from Pseudomonas aeruginosa LP602 exhibited maximum lipase activity at pH 8.0 where it was also stably maintained. At 55 °C, the lipase had the highest activity but not stability (Dharmsthiti and Kuhasuntisuk, 1998). Umesh et al. (2003) reported that lipase from Pseudomonas mendocina PK-12CS was stable at room temperature for more than a month and expressed maxi- FIG. 5 - Effect of pH on the activity of lipase from Burkholderia mum activity at 37 °C and pH 8.0. Gao et al. (2000) reported cepacia LP08. that lipase from Pseudomonas had maximum activity at 45 °C and pH 9.0. Kulkarniand Gadre (2002) reported that lipase from Pseudomonas fluorescens NS2W had an optimal activity at 55 °C and at pH 9.0. Lin et al. (1996) reported that lipase from Pseudomonas pseudoalcaligenes F-111 was stable in the pH range of 6 to 10, and exhibited highest activities at 40 °C. There is no report about Burkholderia produces the low-temperature and alkaline lipase as we know so far. Besides optimal temperature and pH are in low-temperature and alkaline range, a good detergent lipase should also be sta- ble in the presence of surfactants. The lipase from Burkholderia cepacia LP08 was stable in the presence of some surfactants. The above finding was similar to that by Umesh et al. (2003) who found that lipase from Pseudomonas mendocina PK-12CS showed appreciably good stability to Triton X-100, Tween-20 and Tween-80. Ruchi et al. (2007) reported that lipase from Pseudomonas aeruginosa retained 100% activity in presence of Triton X-100, Tween-20 and Tween-80. Rathi et al. (2001) reported that the lipase from Burkholderia cepacia retained 93, 57, 40 and 53% of the activity in the presence of Triton X-100, Tween-20, Tween-80 and saponin respectively. On the other hand, Karadzic et al. (2006) reported lipase from Pseudomonas FIG. 6 - pH stability of lipase from Burkholderia cepacia LP08. aeruginosa was strongly stimulated by Triton X-100 and Tween- Ann. Microbiol., 59 (1), 105-110 (2009) 109 TABLE 2 - Lipase stability in presence of the surfactants, component of detergents, oxidizing agents and protease Surfactants / Protease Relative Detergents/Oxidizing agents Relative (1% w/v or v/v) activity (%) (1% w/v or v/v) activity (%) Control 100 Control 100 Surfactants 1% TritonX-100 69 Component of 4% Glycerine 209 detergents 0.1% TritonX-100 90.3 0.4% Glycerine 281 1% Tween-20 52.9 4% NaCl 266 0.1% Tween-20 91.6 0.4% NaCl 244 1% Tween-80 58.4 3% Borax 77.4 0.1% Tween-80 93.3 0.3% Borax 287 1% SDS 18.5 2% Sodium citrate 116 0.1% SDS 60.4 0.2% Sodium citrate 167 1% Saponin 53 Oxidizing agents 1% Hydrogen peroxide 91 0.1% Saponin 84 0.1% Hydrogen peroxide 93 1% Sodium cholate 131.5 1% Sodium perborate 95 0.1% Sodium cholate 125.2 0.1% Sodium perborate 96 1% Sodium taurocholate 177.9 1% Sodium hypochlorite 92 0.1% Sodium taurocholate 138.3 0.1% Sodium hypochlorite 95 Alkaline protease 0.1% 88.4 Commercial 1% 134 detergent (The 0.01% 100 0.1% 104 brand of bilang) 80. 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Annals of Microbiology – Springer Journals

Published: Nov 24, 2009

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